In the T cell receptor (TCR) gene therapy, of which application mainly to specific cancers is being investigated, a cancer antigen-specific TCR gene is introduced into lymphocytes of a cancer patient. In order to obtain an antigen-specific TCR gene, it is necessary to specify a T cell that can recognize the cancer antigen from T cells among peripheral blood lymphocytes (PBL) collected from the patient, and to clone the TCR gene. Approaches generally made for this purpose include establishing an antigen-specific T cell clone, and this procedure usually requires several months. However, the inventors of the present invention developed a method for amplifying a gene from a single cell by using the 5′-RACE method, which does not require establishment of a cell clone (Patent document 1).
The inventors of the present invention also constructed a system that can detect a cancer-specific T cell from peripheral blood T lymphocytes of a cancer patient, amplify cDNA of TCR that recognizes cancer from a single cancer-specific T cell, and verify the cancer-specificity thereof within ten days in the shortest case (Patent document 2, Non-patent document 1). In this method, the aforementioned 5′-RACE method was used for amplifying a target cDNA.
Amplification of a TCR gene and one-step expression cloning have also been proposed by Moysey et. al. (Non-patent document 1).